PROJECT SUMMARY/ABSTRACT Enzymes are remarkable nanomachines that play a myriad of essential functions in cellular metabolism. Modulation of enzyme structure and flexibility by cofactor/substrate binding provides an important source of regulation of enzyme function, yet our understanding of the fundamental mechanisms coupling protein dynamics to enzymatic activity is still largely incomplete. Indeed, while our appreciation of how conformational dynamics mediate biological function is predominantly based on structural studies on low-complexity, low- molecular weight systems, enzymes are typically oligomeric, multidomain proteins whose biological function depends on an intricate coupling among intradomain, interdomain, and intersubunit conformational equilibria. Without a comprehensive, atomic-resolution understanding of conformational dynamics-mediated, self- regulatory mechanisms in high-complexity, high-molecular weight enzymes, our ability to understand and exploit ubiquitous phenomena in biology, such as allosterism and cooperativity, will continue to lag. Here, we will use NMR combined with other biophysical and biochemical approaches to reveal how the complex interplay between cofactor/substrate binding and conformational dynamics regulates the activity of high molecular weight enzymes that are essential for human and bacterial metabolism. The systems of interest in this proposal are Enzyme I (EI) of the bacterial phosphotransferase system (PTS), and the human RNA demethylases FTO and Alkbh5. EI is a 128 kDa dimeric enzyme whose activity depends on the synergistic action of four conformational equilibria that results in a series of large intradomain, interdomain, and intersubunit structural rearrangements modulated by substrate binding. The PTS is a central regulator of bacterial metabolism that controls multiple cellular functions, including virulence and biofilm formation, through phosphorylation-dependent protein-protein interactions. Therefore, understanding EI activity at atomic level will illuminate the fundamental mechanisms governing long-range interdomain communication in proteins, and may suggest new therapeutic strategies to combat bacterial infections. The second part of the present proposal focuses on enzymes that are capable of catalyzing oxidative demethylation of the N6-methyladenosine (m6A). m6A is the most abundant modification in eukaryotic mRNA. Dynamic regulation of the m6A modification plays an important role in gene expression, cellular response to external stimuli, oncogenesis, adipogenesis and in development of other human diseases. We will investigate the mechanisms that regulate the function of the human RNA demethylases FTO and Alkbh5 with atomic resolution. Our results will guide new strategies to achieve selective inhibition of FTO and Alkbh5 to control gene expression and to contrast progression of cancer. In summary, my research program will elucidate the coupling between large scale conformational changes and function in two distinct classes of high molecular weight multidomain enzymes, providing new insights for future therapies for obesity and cancer as well as novel antibiotic targets.